Enzyme thermostable

Dual-Enzyme Processes. In some cases, especially in symp production in Europe, a Hquefaction process is used that incorporates both a thermostable enzyme and a high temperature heat treatment. This type of process provides better hydrolyzate tilterabiHty than that attained in an acid Hquefaction process (9). Consequendy, dual-enzyme processes were developed that utilized multiple additions of either B. licheniformis or B. stearothermophilus a-amylase and a heat treatment step (see Eig. 1). 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

The research group of Backvall employed the Shvo s ruthenium complex (1) [21] for the racemization. This complex is activated by heat. For the KR they used p-chlorophenyl acetate as the acyl donor in combination with thermostable enzymes, such as CALB [20] (Figure 4.7). This was the first practical chemoenzymatic DKR affording acetylated sec-alcohols in high yields and excellent enantioselectivities. In the best case 100% conversion (92% isolated yield) with 99% ee was obtained. This method was subsequently applied to a variety of different substrates and it is employed (with a different ruthenium complex) by the Dutch company DSM for the large-scale production of (R)-phenylethanol [22]. 

The thermostable enzyme PAMO was the first BVMO identified to oxidize enolizable diketones in acceptable stereoselectivity (82% ee). The J -acetate obtained was hydrolyzed to J -hydroxyphenylacetone as an interesting intermediate for various pharmaceutical compounds (Scheme 9.23) [179]. 

All these capabilities enable to address the search for new enzymes for specialty applications, such as thermostable and solvent resistance enzymes. A viscosity improving thermostable enzyme family (Pyrolase 160 and Pyrolase 200, the later working at higher temperatures) was designed for use in deep and high temperature oil wells. Sales of this product commenced in January 1999, but were discontinued later. 

Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

In this chapter we describe the use of pea seeds to express the bacterial enzyme a-amylase. Bacterial exoenzymes like the heat stable a-amylase from Bacillus licheni-formis are important for starch hydrolysis in the food industry. The enzymatic properties of a-amylase are well understood [13,14], it is one of the most thermostable enzymes in nature and it is the most commonly used enzyme in biotechnological processes. Although fermentation in bacteria allows highly efficient enzyme production, plant-based synthesis allows in situ enzymatic activity to degrade endogenous reserve starch, as shown in experiments with non-crop plants performed under greenhouse conditions [12,15]. Finally, the quantitative and sensitive detection of a-amylase activ-... 

Schulz, F., Leca, F., Hollmann, F. and Reetz, M.T., Towards practical biocatalytic Baeyer-Villiger reactions applying a thermostable enzyme in the gram-scale s3mthesis of optically-active lactones in a two-liquid-phase system. BeilsteinJ. Org. Chem., 2005,1, 10. 

In particular, thermostable enzymes (thermozymes), which are optimally active between 60 and 125 °C, have attracted increasing attention in the recent... 

The major current industrial use for saccharidases is in the manufacture of high fructose com syrup from starch. The current process requires thermostable enzymes and three processing steps because the enzymes used are not compatible at the same pH and temperature (see Figure 1). Industry is looking to improve the process by developing 1) an improved a-amylase that works at low pH that has a low Ca" ... 

Fusions to thermostable enzymes will allow us to evaluate adsorption at higher temperatures. When a column containing Abg-CBDcex/ adsorbed to cellulose at pH 7.0, was eluted with an increasing or decreasing pH gradient (constant ionic strength), protein (enzymatically inactive) was eluted above pH 9, but there was no desorption evident at low pH. (Ong, E. Gilkes, N.R Miller, R.C., Jr. Warren, R.A.J. Kilbum, D.G. Enzyme Microb, TechnoL, in press). 

Thermostability of Thermoanaerohacter sp. CGTase. The addition of 40ppm Ca+ + to the CGTase preparation during incubation at high temperatures in the absence or presence of starch substrate provided no enhancement of the thermostability of the enzyme. A comparison of the thermostable CGTase was made to other thermostable enzymes used in starch liquefaction including Termamyl Bacillus licheniformis) and Bacillus stearothermophilus alpha-amylase. 

Chapters 1 through 6 outline our understanding of the enzymes necessary or potentially useful for biomass conversion. Included are chapters on fuels and chemical feedstock production, pulp and paper processing, waste processing and degradation, food processing, and specific classes of alkali or thermostable enzymes. 

It has been shown possible to convert gaseous substrates with enzymes (Russell and Yang, 1996 Lamare and Legoy, 1993). The enzyme is present as a solid phase which is passed by a stream of gaseous substrate. There is no need for a liquid phase. An example of such a reaction is the oxidation of ethanol by alcohol oxidase. Relatively high reaction temperatures are used to keep the substrates in the gas phase. Consequently, it is favourable to use thermostable enzymes for these applications. 

In order for the reaction to occur with an acceptable rate it is normally required that at least part of the substrates are present in a liquid form (or gaseous, for gas phase reactions). In many cases this is easily achieved but for high-melting substrates there is often a need for elevated temperatures, which makes it necessary to use thermostable enzymes. In some cases the substrates can be chosen so that they form an eutectic mixture, which is characterised by a considerably lower melting point than the individual substrates. In some case the melting point has been further lowered by the addition of small amounts of extra components, often polar organic solvents (Gill and VuUson, 1994). 

Kim and Park subsequently reported that ruthenium pre-catalyst 2 racemizes alcohols within 30 min at room temperature [53]. However, when combined with an enzyme (lipase) in DKR at room temperature, very long reaction times (1.3 to 7 days) were required, in spite of the fact that the enzymatic KR takes only a few hours (Scheme 5.24). Despite these compatibility problems, their results constituted an important improvement, since chemoenzymatic DKR could now be performed at ambient temperature to give high yields, which enables non-thermostable enzymes to be used. More recently, we communicated a highly efficient metal- and enzyme-catalyzed DKR of alcohols at room temperature (Scheme 5.24) [40, 54]. This is the fastest DKR of alcohols hitherto reported by the combination of transition metal and enzyme catalysts. Racemization was effected by a new class of very... 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

Polymerase Chain Reaction DNA Amplification with a Thermostable Enzyme... 

It is practical to use PCR reagents in 10 x working solutions that will be diluted by a factor of ten for final concentration in the PCR samples. An exception is the thermostable enzyme Taq polymerase, which is stored at a much higher concentration. All reagents are available from numerous companies. 

Bock, A.K. Glasemacher, J. Schmidt, R. Schoenheit, P. Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima. J. BacterioL, 181, 1861-1867 (1999)... 

Ramon-Maiques, S. Marina, A. Uriarte, M. Fita, L Rubio, V. The 1.5 A resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic Archaeon pyrococcus furio-sus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases. J. Mol. Biol., 299, 463-476 (2000)... 

For PCR, it is important to use a licensed thermostable enzyme with a proofreading facility and to reduce the cycle number to the minimum number that provides acceptable yield after the post-PCR manipulations. The precautions are necessary, because the error rate for some thermostable polymerases is quite high and the number of errors that accumulate increases with cycle number. 

Zhang Z, Tsujimura M, J-i A et al (2005) Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. J Biol Chem 280 9698-9705... 

The key to the successful scale-up was the immobilization of the enzyme, which increased stability and reduced the enzyme costs to an acceptable level. The isomerization step is typically carried out in a parallel series of packed-bed reactors, where the enzyme is immobilized on silica or inert cellulose carriers (Figure 5.22). A ton of immobilized xylose isomerase can catalyze the production of 5000 tons of HFCS [35]. Current research is concentrating on developing a more thermostable enzyme variant which would reach the 5 5 45 ratio directly in the reactor column. 

E Keinan, EK Hafeli, KK Seth, RR Lamed. Thermostable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Ther-moanaerobium brockii. J Am Chem Soc 108 162-168, 1986. 

K Imada, M Sato, N Tanaka, YKatsube, Y Matsuura, T Oshima. Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. J Mol Biol 222 725-738, 1991. 

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